Ergot cyclic peptide alkaloids having a venotonizing effect, useful for treating orthostatic hypotension

ABSTRACT

9-thia ergot cyclic peptide alkaloids produced by fermentation or synthetic methods have interesting anti-parkinson and other pharmacological activities.

This is a division of application Ser. No. 751,272, filed July 2, 1985,now U.S. Pat. No. 4,681,880, which in turn is a division of applicationSer. No. 404,832, filed Aug. 3, 1982, now U.S. Pat. No. 4,542,135.

This invention relates to ergot cyclic peptide alkaloids, theirproduction and pharmaceutical compositions containing them.

Such ergot cyclic peptide alkaloids contain an optionally substitutedergoline nucleus linked at the 8-position thereof to a tricyclic peptidemoiety commonly referred to as an amino-cyclol, e.g.2-amino-octahydro-10b-hydroxy-3,6-dioxo-8H-oxazolo[3,2-a]pyrrolo[2,1-c]pyrazineand analogues thereof.

Such ergot alkaloids may be obtainable from natural sources, e.g. byfermentation, and by chemical synthetic methods, or be obtainable onlyby chemical synthetic methods.

The present invention provides in one aspect 9'-thia ergot cyclicpeptide alkaloids, herein referred to as compounds of the invention.

The methylene group in the 9 position of the aminocyclol is accordinglyreplaced by sulphur. Such ergot cyclic peptide alkaloids may exist inisomeric form. For example in the 8 position of the ergoline nucleus thecarbon atom may have the R or S configuration.

In particular the present invention provides a compound of formula I##STR1## wherein R₁ is (C₁₋₄)alkyl,

R₂ is (C₁₋₆)alkyl or benzyl,

R₃ and R₄ independently are hydrogen or (C₁₋₄)alkyl,

R₅ is hydrogen or bromine,

R₆ and R₇ are each hydrogen, or

R₆ and R₇ together form a single bond, or

R₆ is methoxy and R₇ is hydrogen, and

R₈ is hydrogen, methyl, or halogen of atomic number from 9 to 35,

with the proviso that when R₅ is bormine, then R₇ is hydrogen.

In formula I R₁ is for example methyl or isopropyl. R₂ is for examplen-or iso-propyl, n-, iso- or sec-butyl, or benzyl. R₃ is for example n-or iso-propyl or preferably ethyl or methyl. R₄ is preferably hydrogenor methyl. R₅ is preferably hydrogen. R₆ and R₇ conveniently form asingle bond or are each hydrogen. R₈ is conveniently hydrogen, bromineor methyl.

As indicated above the compounds of formula I may exist in isomericform, e.g. the 8R and 8S isomers. The 8R isomers are preferred.

In accordance with the present invention a compound of the invention maybe produced by a process characterised by

(a) condensing an acid addition salt of an appropriate9-thia-aminocyclol or a precursor thereof with an reactive acidderivative of an appropriate lysergic acid derivative or a precursorthereof, or

(b) for the production of a 9'-thia analogue of a ergot cyclic peptidealkaloid having a 9'-methylene group and obtainable by fermentation,cultivating a strain capable of producing the 9'-methylene ergot cyclicpeptide alkaloid in the presence of L-thiazolidine-4-carboxylic acid ora precursor thereof.

The 9'-thia ergot cyclic peptide alkaloid may be recovered e.g. in freebase form or in acid addition salt form.

In particular a compound of formula I as defined above may be producedby a process characterised by

(a) condensing an acid addition salt of a compound of formula II##STR2## wherein R₁ and R₂ are as defined above, or a precursor thereof,with a reactive acid derivative of a compound of formula III ##STR3##wherein R₃, R₄, R₅, R₆, R₇ and R₈ are as defined above or a precursorthereof, or

(b) for the production of a compound of formula Ia ##STR4## wherein R₁^(I) is methyl and R₂ ^(I) is isobutyl or benzyl or

R₁ ^(I) is ethyl and R₂ ^(I) is benzyl, or

R₁ ^(I) is isopropyl and R₂ ^(I) is isopropyl, sec-butyl, isobutyl, orbenzyl,

cultivating a strain capable of producing the corresponding ergotalkaloid having a 9'-methylene group in the presence ofL-thiazolidine-4-carboxylic acid or a precursor thereof.

The compound of formula I or Ia may be recovered, e.g. in free base formor in acid addition salt form.

As indicated above a precursor of a particular starting material may beused.Such a precursor may be a compound which is capable of beingconverted into the starting material using conventional reactions. Afterthe above defined step (a) or (b) is effected then the product may beconverted into a compound of the invention, e.g. using the conventionalreactions. For example an amino group may be temporarily protected.

Process (a) may be effected in conventional manner for the production ofergot cyclic peptide alkaloids by condensing appropriate aminocyclolswith appropriate lysergic acid derivatives.

Conveniently the aminocyclol is in the form of the hydrochloride. Thelysergic acid derivative is conveniently the acid chloride, acidazide,or a mixed anhydride with sulphuric acid or trifluoroacetic acid.Preferably the lysergic acid derivative is an addition product withdimethylformamide or acetamide, and thionyl chloride, phosgene, oroxalyl chloride. Preferably the reaction is effected in the presence oftriethylamine or pyridine. Suitable solvents are for example chloroform,methylene chloride, dimethylformamide, or acetonitrile.

Suitable reaction temperatures are from about -30° to about +20° C.

The 9-thia-aminocyclol compounds used as starting materials are new andalso form part of the invention.

The preferred 9-thia-aminocyclols are those of formula II as definedabove.

The compounds of formula II may be produced in conventional manner asdescribed in the following flowsheet wherein Y is a protecting group,e.g. ethoxy, and Z is a protecting group, e.g. benzyloxycarbonyl. Other9-thia-aminocyclols may be produced in analogous manner. ##STR5##

The compounds VI, VII, VIII, IX and X are new and each per se forms partof the present invention.

The lysergic acid derivatives are in general known.

Process (a) is the preferred process for the production of the compoundsof the invention.

Process (b) may be effected in conventional manner for the protection ofergot cyclic peptide alkaloids by cultivation of microorganisms.

The strains used for cultivation may be strains used for the productionof the corresponding ergot cyclic peptide alkaloids having a methylenegroup in the 9'-position, preferably ergotamine and ergocristine. Suchstrains may be strains of Claviceps purpurea. The strains may beisolated from nature or produced from other Claviceps purpurea strainswhich are treated with radiation or mutagenic substances, or produced byselection from other strains.

For the production of 9'-thia-ergotamine and 9'-thiaergotaminine thepreferred strain is the ergotamine/ergotaminine producing strain ofClaviceps purpurea deposited on Sept. 20, 1979 at the U.S. Department ofAgriculture (Northern Regional Research Laboratory), Peoria, Ill.,U.S.A. under the number NRRL 12043. For the production of9'-thia-ergocristine and 9'-thiaergocristinine the preferred strain isthe ergocristine/ergocristinine producing strain of Claviceps purpureadeposited on Sept. 20, 1979 at the U.S. Department of AgriculturePeoria, Ill. U.S.A., unter the number NRRL 12044.

These strains are new and form part of the present invention

The strains NRRL 12043 and 12044 are freely available from theabove-mentioned depository and also from the patentee.

Suitable strains used for the production of other ergot cyclic peptidealkaloids are known and are freely available from established sources.

Characterisation and fermentation of strains NRRL 12043 and NRRL 12044(a) Characterisation of the strain NRRL 12043

The strain used as starting material was isolated from an ergotaminecontaining sclerotium obtained from Bromus, Valais, Switzerland and wassubjected to several mutation steps using ultra-violet radiation andmutagenic chemicals to produce NRRL 12043.

A small mycelium piece of the strain is inoculated in the centre of anAgar plate of a sterilized medium.This has the following composition: 60g saccharose, 10 g asparagine, 0.5 g caesin amino acids (known under thebrand Difco), 0.25 g KH₂ PO₄, 0.25 g MgSO₄.7H₂ O, 0.125 g KCl, 16 mgFeSO₄.7H₂ O, 10 mg ZnSO₄.7H₂ O, 15 g Agar and distilled water to 1liter. The pH is adjusted to 6.0 with 25% ammonium hydroxide. The mediumis sterilized at 120° C. for 20 minutes.

After 7 days at 24° C. a colony of about 3 cm in diameter is produced.After 14 days the diameter is about 5 cm and after 14 days about 8 cm.The colony is elevated and slightly wrinkled. The edge is diffuse. Thecolour of of the colony is beige-violet in the centre and at the edgegrey-beige. A 20 day old colony contains per cm² about 1×10⁸ conidia.The conidia are oval. Their dimensions about 6-12×4-7 microns.

(b) Characterisation of the strain NRRL 12044

The strain used as starting material was isolated from an ergocristinecontaining sclerotium obtained from Roggen, N.America and was subjectedto several mutation steps using ultra-violet radiation and mutagenicchemicals to produce NRRL 12044.

A small mycelium piece of the strain is inoculated in the centre of anAgar plate of a sterilized medium.This has the following composition: 70g malt extract (obtained from Wander, Switzerland), 30 g potato extract(Brand Stockli, obtained from Knorr), 15 g Agar and distilled water to 1liter. The pH value is about 5.3 to 5.7. The medium is sterilized at120° C. for 20 minutes.

After 7 days at 24° C. a colony of about 2 cm in diameter is produced.After 14 days the diameter is about 4 cm and after 20 days about 5 cm.

The mycelium is smoothly elevated and covered with white wool-likehyphae, with a plaited structure which towers about it. In the centrethere is a crater-forming hump about 1 cm diameter from which radialplaits extend. The mycelium lying on the agar is coloured violet, andcomprise concentric alternating zones, some strongly coloured and othersless strongly coloured, the edge being diffuse. A 20 day old colonycontains per cm² about 1×10⁸ conidia. These vary a lot in theirdimensions, 5-12×3-7 microns, but mostly are 5-6×3 microns.

The mycelium contains no alkloids.

Cultivation and inoculation (a) NRRL 12043

Cultivation and inoculation of the strain NRRL 12043 may effected bywashing the conidia from one of the above described colohies in sterilewater and then inoculating for example an agar sterilized medium in aagar-slant. This comprises 70 g malt extract (brand Wander,Switzerland), 30 g potato extract (brand Stockli, Knorr, Switzerland),15 g Agar and distilled water to 1 liter. The pH value may be from 5.3to 5.7. The medium may be sterilized for 20 minutes at 120° C. After 14days such a agar-slant culture containing 12 ml medium in a glass tubeof 18 mm diameter and 20 cm in length has about 1.10⁹ conidia. Theconidia from such a agar slant are washed in sterile water to produce aconcentration of about 10⁶ -10⁷ conidia per ml. 0.5 ml of thissuspension is used to inoculate agar slants having the same agar medium.These cultures are cultivated at 24° C. for 14 days and then stored at-40° C.

(b) NRRL 12044

Cultivation and inoculation of the strain NRRL 12044 may be effected bywashing the conidia from one of the above described colonies in sterilewater and then inoculating for example the above defined agar medium forNRRL 12044. After 18 days such a agar slant culture grown as for NRRL12043 contains about 1-2.10⁹ conidia These conidia are furthercultivated as for NRRL 12043.

Pre-culture

Preferably a preculture of the strain is made before the fermentationculture,using a medium which allows good spore cultivation and a quickmycelial growth. Suitable media may be concentrated carbon sources inthe form of a mono or di-saccharide, optionally in combination with apolyalcohol, a nitrogen source in the form of an amind acid or anammonium salt, mineral salts, trace elements and plant additives.

Preferably the pH is from 5 to 7. Firstly the medium may be adequatelyinoculated with conidia and incubated in shaker machines or fermentersat about 22° to 26° C. for 4 to 7 days. A thick culture results ofloose, woollike unpigmented mycelial pieces sized about 2 mm to 3 mmwhich have long hyphae of about 2 to 6 microns in diameter.

Fermentation culture

The fermentation culture may be inoculated by part of the pre-culture,or the last of a series of pre-cultures, in a nutrient medium whichpreferably has a composition which provides in a short time a highmycelium mass.

Suitable carbon sources include saccharose; suitable nitrogen sourcesinclude ammonium salts such as the oxalate, citrate, succinate orformate. Mineral salts and trace elements such as iron and zinc shouldbe present. The cultures may be cultivated in Erlenmeyer flasks orfermenters. An adequate air supply should be present. The preferredtemperature is 24° C.

At the start of the alkaloid formation, generally between the 3rd and6th day of the fermentation the sulphur source may be added, e.g.L-thiazolidine-4-carboxylic acid in free form or in protected form or insalt form e.g. the potassium, sodium or ammonium salt. The preferredconcentration of this compound is about 1 to 5 g/liter culture.

The culture is preferably cultivated for a further 6 to 12 days. Within9 to 18 days a thick culture which mainly comprises besides finemycelial pieces mainly compact cylindrical mycelial pieces which areabout 0.5 to 3 mm long and about 0.1 to 1 mm thick. The mycelial piecesappear like plectenchymic tissue of spherical or polyhedric toshort-cylindrical, thick walled and strongly vaculated cells of6-12×6-14 microns, mainly 8×9 microns, which are traversed by up to 100micron long hyphae. They have a brown pigment. The culture filtrate isalso coloured brown.

When the increase in weight and in the alkaloid content of the myceliumlessens the alkaloids may be extracted in conventional manner withorganic solvents from the whole culture broth.Alternatively the myceliummay be separated by filtration or centrifugation and the mycelium andthe non-mycelium part separately extracted.The ergot alkaloids existmostly in the mycelium.

Isolation of 9'-thia ergot cyclic peptide alkaloids

These alkaloids may be isolated from the culture broth,the culturefiltrate or the mycelium using conventional extraction methods. Thepurification may be effected using known purification methods, e.g.using different solvents or converting the substances into difficultlysoluble salts. Preferably purification is effected bychromatography,e.g. on aluminium oxide, silicagel, sephadex etc usingappropriate solvent systems. Preferably the isolation is effected in thedark,and minimizing formation of the free base form and the use of polarsolvents in order to minimize decomposition or isomerisation.

In another aspect the invention provides a biologically pure or axemicClaviceps purpurea culture having the identifying characteristics of thestrain deposited under the accession number NRRL 12043 or 12044.

In a further aspect the invention provides a culture medium containing astrain or culture as defined above.

If desired the compounds of the present invention may be converted intoother compounds of the invention e.g. using known methods forinterconversion of ergot cyclic peptide alkaloids. Conveniently the thiaergot cyclic peptide alkalbid is alkylated and/or hydrogenated toproduce an alkylated and/or hydrogenated thia ergot cyclic peptidealkaloid.

For example the compounds may be alkylated in position 1 or 2 of theergoline nucleus using appropriate selective alkylation methods. Ifdesired any 9,10 double bond in the ergoline nucleus may be satutatede.g. by catalytic hydrogenation to give the corresponding9,10-dihydro-9'-thia ergot cyclic peptide alkaloids. The compounds ofthe invention may be isolated and purified in conventional manner. Thecompounds of the invention may be produced as a mixture of the 8R and 8Sisomers especially when a double bond is present in the 9,10 position.These may be separated by chromatography. If desired the 8R and 8Sisomers may be epimerized in known manner, e.g. by treatment with acids.

Free base forms of the compounds of the invention may be converted intoacid addition salt forms in conventional manenr and vice versa. Suitableacids for salt formation include hydrochloric acid, sulphuric acid,maleic acid, fumaric acid, tartaric acid and methanesulphonic acid.

Insofar as the production of any particular starting material is notparticularly described, the compound is known or may be made inconventional manner.

In the following examples all temperatures are in degrees Centigrade andare uncorrected.

EXAMPLE 1 [Process a] 9,10dihydro-9'-thia-α-ergocryptine or N-([2R,5S,10aS,10bS]-hexahydro-10b-hydroxy-5-isobutyl-2-isopropyl-3,6-dioxo-8H,10H-oxazolo[3,2-a]thiazolo[4,3-c]pyrazin-2-yl)-6-methyl-5R-5β,8β)ergoline-8-carboxamide

30 ml of absolute dimethylformamide are cooled with stirring to -15° anda solution of 1 ml oxalyl chloride in 5 ml absolute acetonitrile isadded dropwise thereto. 2.95 g dry 9,10-dihydrolysergic acid are addedand a grey-white precipitate is formed. The mixture is stirred for 30minutes, treated at 0° with 6 ml absolute pyridine with vigorouscooling, and then with 1.40 g (2R,5S,10aS,10bS)-2-amino-dihydro-10b-hydroxy-5-isobutyl-2-isopropyl-8H,10H-oxazolo[3,2-a]thiazolo[4,3-c]pyrazin-3(2H),6(5H)-dionehydrochloride, and then stirred vigorously for 2 hours. The temperatureof the mixture is allowed to rise slowly from -10° to 0°.

To work up, 6.5 ml citrate buffer (pH 4) is added to the mixture withstrong cooling. The mixture is made alkaline with 2N sodium carbonatesolution, and extracted thrice with methylene chloride. The combinedextracts are dried over sodium sulphate, filtered and concentrated. Theconcentrate is chromatographed on 100 g silicagel to afford on elutionwith 3% methanol in methylene chloride, the title compound which iscrystallized from methylene chloride/ether.

M.pt.: 182°-184° (decomp); [α]_(D) ²⁰ =+9.6° (c=0.5 indimethylformamide).

The amino cyclol is obtained as follows:

(a)(6S,8aS)-dihydro-6-isobutyl-3H-thiazolo[3,4-a]-pyrazin-5(6H),8(7H)-dione

61 g L-thiazolidine-4-carboxylic acid methyl ester in 450 ml absoluteether are added to a solution of 95.8 gN-tert-butyloxycarbonyl-L-leucine in 450 ml absolute ether. The mixtureis stirred for 15 minutes at room temperature. A solution of 94.4 gdicyclohexylcarbodiimide in 200 ml absolute ether is added dropwise andafter being stirred for 90 minutes the mixture is filtered to remove theurea. The filtrate is extracted in turn with 1N hydrochloric acid, icewater, 2N sodium bicarbonate solution and ice water.

The aqueous phases are back-extracted with ether. The organic phases arecombined, dried with sodium sulphate, filtered and concentrated. Theresultant residue is reacted further as such.

The so-obtained protected dipeptide is dissolved in 350 ml absolutemethylene chloride and the solution is treated with 140 mltrifluoroacetic acid with ice cooling. The mixture is maintainedstanding at room temperature for 16 hours and then concentrated in arotary evaporator. The residue is taken up in methylene chloride andmade alkaline with 2N sodium carbonate solution. The mixture is twiceextracted back with methylene chloride. The combined organic phases areconcentrated, dried over sodium sulphate, filtered and concentrated. Theheading compound crystallized after taking up in ether and addinghexane.

M.pt. 142°-143°; [α]_(D) ²⁰ =113° (c=0.8 in chloroform).

(b) (2R,5S,10aS,10bS)-Hexahydro-10b-hydroxy-5-isobutyl-2-isopropyl-3,6-Hdioxo-8H,10H-oxazolo[3,2-a]thiazolo[4,3-c]pyrazine-2-carboxylic acidethyl ester

72.7 g(6S,8aS)-Dihydro-6-isobutyl-3H-thiazolo[3,4-a]-pyrazin-5(6H),8(7H)-dionein 175 ml absolute dioxane are treated with 114.5 gS(+)-isopropyl-benzyloxy malonic acid mono ethyl ester acid chloride and50.9 g 2,6-lutidine. The mixture is stirred for 4 hours at 70°, dilutedwith 1 liter ether and treated twice with in turn 2N hydrochloric acidand saturated sodium bicarbonate solution. The aqueous phases areback-extracted twice with ether. The combined ether phases are driedover sodium sulphate, filtered and concentrated in a vacuum. The brownresidue is chromatographedon 2.4 kg silicagel to afford, onelution with2% methanol in methylene chloride, the resultant acyl derivative whichis further reacted as such after concentration to give a yellow oil.

The resultant acyl derivative is dissolved in 600 ml trifluoroaceticacid. The mixture is allowed to stand for 18 hours at room temperature.The resultant red-brown solution is concentrated. The residue is takenup in 1 liter methylene chloride and made alkaline with 2N sodiumcarbonate solution. The aqueous phase is separated off andback-extracted twice with 300 ml methylene chloride. The combinedorganic phases are dried over sodium sulphate, filtered and concentratedin a vacuum. The red brown crude product is chromatographed on 2.4 kgsilicagel to give on elution with 2% methanol in methylene chloride theheading compound which is crystallized from ether/petroleum ether togive white crystals.

M.pt. 106°-108°; [α]_(D) ²⁰ =-7.5° (c=1.0 in chloroform).

(c)(2R,5S,10aS,10bS)-hexahydro-10b-hydroxy-5-isobutyl-2-isopropyl-3,6-dioxo-8H,10H-oxazolo[3,4-a]thiazolo[4,3-c]pyrazine-2-carboxylicacid

27.6 g(2R,5S,10aS,10bS)-hexahydro-10b-hydroxy-5-isobutyl-2-isopropyl-3,6-dioxo-8H,10H-oxazolo[3,2-a]thiazolo[4,3-c]pyrazine-2-carboxylicacid ethyl ester are dissolved in 600 ml 1N sodium hydroxide solutionand stirred for 5 hours at room temperature. To work up the mixture isadjusted to pH 4.5 with 2N hydrochloric acid and repeatedly extractedwith ethyl acetate. The organic extracts are dried, and concentrated atunder 30° to remove the solvent. The heading compound is obtained aswhite crystals on dilution with ether.

M.pt. 149°-151° (decomp).

(d)(2R,5S,10aS,10bS)-Hexahydro-10b-hydroxy-5-isobutyl-2-isopropyl-3,6-dioxo-8H,10H-oxazolo[3,2-a]thiazolo-[4,3-c]pyrazine-2-carbonyl-azide

A solution of 6 ml oxalyl chloride in 50 ml absolute methylene chlorideis added dropwise within 10 minutes to a stirred mixture of 7 mlabsolute dimethylformamide and 100 ml absolute methylene chloride cooledto -15°. The mixture is stirred for a further 10 minutes, and dilutedwith 100 ml absolute ether. 17.3 g (2R,5S,10aS,10bS)-Hexahydro-10b-hydroxy-5-isobutyl-2-isopropyl-3,6-dioxo-8H,10H-oxazolo[3,4-a]thiazolo[4,3-c]pyrazin-2-carboxylicacid are quickly added. A clear solution results which is stirred for 10minutes at -10°. A solution of 30 g sodium azide in 120 ml water isadded. The mixture is treated with 800 ml methylene chloride and theresultant two phase mixture is vigorously shaken for 4 minutes. 1 literice cold saturated sodium bicarbonate solution is added. The organicphase is separated off and dried over sodium sulphate. The solvent isremoved by concentrating the mixture to give a residue which ontreatment with absolute ether yields heading compound as white crystals.

M.pt. 100° (explosion).

(e) (2R,5S,10aS,10bS)-2-Amino-dihydro-10b-hydroxy-5-isobutyl-b2-isoproply-8H,10H-oxazolo[3,2-a]thiazolo[4,3-c]pyrazin-3(2H),6(5H)-dione

A solution of 12.7 g(2R,5S,10aS,10bS)-hexahydro-10b-hydroxy-5-isobutyl-2-isopropyl-3,6-dioxo-8H,10H-oxazolo[3,2-a]thiazolo[4,3-c]pyrazin-2-carbonylazide in 180 ml absolute methyl ethyl ketone isttreated with 3.18 ml 10Nhydrochloric acid solution and boiled under reflux for 15 minutes. Theresultant mixture is concentrated, and diluted with absolute ether togive the heading compound in hydrochloride salt form as yellow crystals.

M.pt. 123°-126° (decomp).

EXAMPLE 2 (Process a) 9'-Thia-α-ergocryptinine and9'-Thia-α-ergocryptine also known asN-([2R,5S,10aS,10bS]-Hexahydro-10b-hydroxy-5-isobutyl-2-isopropyl-3,6-dioxo-8H,10H-oxazolo[3,2-a]thiazolo[4,3-c]pyrazin-2-yl)9,10-didehydro-6-methyl-5R-(5α,8α)ergoline-8-carboxamide andN-([2R,5S,10aS,10bS]-Hexahydro-10b-hydroxy-5-isobutyl-2-isopropyl-3,6-dioxo-8H,10H-oxazolo[3,2-a]thiazolo[4,3-c]pyrazin-2-yl)9,10-didehydro-6-methyl-5R-(5β,8β)ergoline-8-carboxamide.

2.5 g anhydrous lysergic acid in 25 ml absolute dimethylformamide aredissolved through the addition of 2.11 g trifluoroacetic acid. Themixture is stirred and cooled to -10° and at this temperature a mixtureof 2.52 g trifluoroacetic acid anhydride in 15 ml absolute acetonitrileis added dropwise within 5 minutes. The clear solution is stirred for 10minutes. Then under vigorous cooling 15 ml pyridine and 2.71 g(2R,5S,10aS,10bS)-2-amino-dihydro-10b-hydroxy-5-isobutyl-2-isopropyl-8H,10H-oxazolo[3,2-a]thiazolo[4,3-c]pyrazin-3(2H),6(5H)-dionehydrochloride are added. The reaction mixture is stirred for a further 1hour from -10° to 0°.

To work up 200 ml methylene chloride are added and the mixture is wellshaken with 100 ml 2N sodium carbonate solution. The aqueous phase isseparated off and is back extracted twice with 100 ml methylene chlorideeach time. The combined aqueous phases are dried over sodium sulphateand concentrated in a vacuum. The residue is chromatographed on 250 gsilicagel to elute with 2% methanol in methylene chloride9'-thia-α-ergocryptinine which is recrystallized from methanol,

M.pt. 228°-230° (decomp); [α]_(D) ²⁰ =+281° (c=0.8 indimethylformamide), and with 3% methanol in methylene chloride firstfractions containing mixtures of ergot alkaloids and then pure9'-thia-α-ergocryptine which is crystallized from ether.

M.pt. 203°-205° (decomp); [α]_(D) ²⁰ =+46° (c=0.7 in dimethylformamide).

EXAMPLE 3 (process b) 9'-thia-ergotamine and 9'-thia-ergotaminine 1.Cultivation of strain NRRL 12043 (a) Pre-culture

About 10⁹ conidia of a slant culture of the Claviceps purpureaergotamine/ergotaminine producing strain NRRL 12043 are suspended in 10ml sterile water.

200 ml of a preculture medium of the following composition:

    ______________________________________                                        Components       g/liter                                                      ______________________________________                                        Saccharose       100                                                          Proflo*          10                                                           Ammonium oxalate 3                                                            Ca(NO.sub.3).sub.2.4 H.sub.2 O                                                                 1                                                            KH.sub.2 PO.sub.4                                                                              0.25                                                         MgSO.sub.4.7H.sub.2 O                                                                          0.25                                                         KCl              0.125                                                        FeSO.sub.4.7H.sub.2 O                                                                          0.0166                                                       ZnSO.sub.4.7H.sub.2 O                                                                          0.0068                                                       distilled water ad                                                                             1 liter                                                      ______________________________________                                         *Proflo is a dried and ground cotton flower seed available from               Pharmamedia, Switzerland having on analysis 60% protein, 4% fat, 22%          sugar, 6% ash, 2% moisture and 6% fibre.                                 

in a 500 ml Erlenmeyer flask are made up. The pH of the mixture isadjusted to 6.5 with ammonium hydroxide. The medium is sterilized at120° C. for 20 minutes. The medium is inoculated with the conidia. Thepre-culture is shaken at 24° on a rotary shaker for 5 days (180 rpm and5 cm shaking circle). At the end of the growth the culture has a pH of5.3 and a dry mycelial mass of the pre-culture of about 22 g/liter.

(b) Fermentation culture

10 ml of the pre-culture is used to inoculate 50 ml of a main culturemedium of the following composition:

    ______________________________________                                        Components       g/liter                                                      ______________________________________                                        Saccharose       240                                                          Ammonium oxalate 9.6                                                          Ca(NO.sub.3).sub.2.4H.sub.2 O                                                                  2.5                                                          MgSO.sub.4.7H.sub.2 O                                                                          0.625                                                        KH.sub.2 PO.sub.4                                                                              0.625                                                        KCl              0.312                                                        FeSO.sub.4.7H.sub.2 O                                                                          0.0252                                                       ZnSO.sub.4.7H.sub.2 O                                                                          0.0102                                                       distilled water ad                                                                             1 liter                                                      ______________________________________                                    

in a 500 ml Erlenmeyer flask. The pH has been adjusted with 25% ammoniumhydroxide to 6.2. The medium is sterilized at 110° for 20 minutes.

The culture is incubated at 24° in a rotary shaker (180 rpm, 5 cmshaking circle). After 4 days cultivation time each culture is treatedwith 90 mg L-thiazolidine-4-carboxylic acid. The cultures are incubatedfor a further 10 days in the rotary shaker. After the fermentation hasfinished the cultures are filtered. The mycelium is dried carefully. Thedry mycelial mass of the main culture is about 85 g/liter. The totalalkaloid content is about 12 mg/g, and the weight of 9'-thiaergotamineand 9'-thiaergotaminine is about 10% of the total alkaloid content.

2. Isolation of 9'-thiaergotamine and 9'-Thiaergotamine

500 g dry mycelium is twice homogenized each time with 1600 ml methanoland 2% concentrated mmonium hydroxide and further twice homogenized eachtime with 1200 ml methanol. each homogenization taking 5 minutes in anultrasonic homogenizer such as an Ultra Turrax apparatus. The combinedfiltrates are concentrated in a vacuum at a maximum of 40° bathtemperature to give about 50 g wine-red residue. The residue isdissolved in 500 ml methanol and filtered through a chromatograph tubeof 10 cm diameter and containing 500 g aluminium oxide of Activity II(Woehlm), which is washed with 2000 ml methanol. The combined filtratesare evaporated to dryness in a vacuum to give 25 g yellow foam. Theresidue is chromatographed on a 200 fold silicagel (Brand 60 Merck)using 2% methanol in methylene chloride and the fractions are examinedby thin layer chromatography and those fractions which have only spotscorresponding to 9'-thia ergot alkaloids are further purified (see tableI).

Table I below gives representative Rf values for ergotamine (Et) andergotaminine (Et-in) and the corresponding thia derivatives in differentthin layer chromatographic systems.

                  TABLE I                                                         ______________________________________                                                     Rf values                                                        Layer and solvent                                                                            Thia-Et  Et      Thia-Et-in                                                                            Et-in                                 ______________________________________                                        Aluminium oxide.sup.(1)                                                       Ethylacetate/sec.Butanol                                                                     0.44     0.34    0.68    0.59                                  9:1                                                                           Toluene/iso-Propanol                                                                         0.58     0.50    0.69    0.63                                  9:1                                                                           Toluene/iso-Propanol                                                                         0.30     0.24    0.58    0.46                                  19:1                                                                          Silicagel.sup.(2)                                                             Toluene/iso-Propanol                                                                         0.15     0.11    0.43    0.31                                  85:15                                                                         CH.sub.2 Cl.sub.2 /MeOH 93:7                                                                 0.25     0.22    0.65    0.52                                  ______________________________________                                         .sup.(1) Aluminium oxide plates Merck F 254 (Type E)                          .sup.(2) Silicagel plates Merck F 254                                    

On the thin layer chromatographic plates the substance spots appearunder UV light (254 nm) as blue-voilet and (366 nm) as bright bluespots. They can also be detected with iodine vapour as brown spots.

The van Urk reagent can also be used to detect the substance, whichappears blue-violet, which is made more intensive in daylight or bytreatment with nitric dioxide.

9'-thiaergotamine

The appropriate chromatography fractions containing only9-thiaergotamine are concentrated, dissolved in ethyl acetate, filteredthrough talc, and after concentration in a vacuum treated with a littlehexane whereupon the alkaloid crystallises out. The crystals arerecrystallized from the same system to give white to yellowish crystals.

M.pt. from 199° (slow decomp) [α]_(D) ²⁰ =-165° (c=0.5 in chloroform)

mesylate m.pt. 211° (decomp); [α]_(D) ²⁰ =+76° (c=0.51 in methanol).

9-thiaergotaminine

In analogous manner to the isolation of 9'-thiaergotamine, theappropriate fractions containing 9'-thiaergotaminine are purified togive white crystals.

M.pt. from 199° (decomp); [α]_(D) ²⁰ =+343° (c=0.55 in chloroform).

EXAMPLE 4 (process b) 9'-thiaergocristine and 9'-thiaergocristinine 1.Cultivation of strain NRRL 12044

The ergocristine/ergocristinine producing Claviceps purpurea strain

NRRL 12044 is cultivated in analogous manner to that described inExample 3. The dry mycelium mass of the main culture is about 80 g/literand the alkaloid content is about 10 mg/g. The title compounds compriseabout 8% of the total alkaloid content.

2. Isolation of 9'-thiaergocristine and 9'-thiaergocristinine

500 g dry mycelia are twice homogenized each time with 1500 ml methanoland 2% concentrated ammonium hydroxide and further twice homogenizedwith 1200 ml ethanol, each homogenization taking 5 minutes in aultra-sonic homogenizer such as an Ultra Turrax apparatus. The combinedfiltrates are concentrated in a vacuum at a maximum 40° bath temperatureto give about 190 g wine-red residue. The residue is dissolved in 900 mlmethanol and filtered through a 10 cm high layer of 600 g aluminiumoxide (basic, activity II) which is washed with 2000 ml methanol. Thecombined filtrates are concentrated to dryness in a vacuum to give aresidue of about 108 g. The residue in methanol is chromatographed on3000 g Sephadex cellulose derivative LH-20 and 200 ml fractionscollected and concentrated. The fractions containing the title thiaergot alkaloids are purified (ca 526 mg dry weight) the Rf values beinggiven in Table II in comparison to ergocristine [Ec] and ergocristinine[Ec-in].

                  TABLE II                                                        ______________________________________                                                     Rf values                                                        Layer and solvent                                                                            Thia-Ec  Ec     Thia-Ec-in                                                                            Ec-in                                  ______________________________________                                        Aluminium oxide.sup.(1)                                                       Ethylacetate/sec.Butanol                                                                     0.68     0.63   0.78    0.70                                   9:1                                                                           Toluene/iso-Propanol                                                                         0.68     0.62   0.69    0.66                                   9:1                                                                           Toluene/iso-Propanol                                                                         0.50     0.42   0.58    0.47                                   19:1                                                                          Silicagel.sup.(2)                                                             Toluene/iso-Propanol                                                                         0.37     0.28   0.56    0.48                                   85:15                                                                         CH.sub.2 Cl.sub.2 /MeOH 93:7                                                                 0.47     0.41   0.70    0.60                                   ______________________________________                                         .sup.(1) and .sup.(2) see Table I. For detection see example 3.          

The fractions containing the thia title compounds are chromatographed onSephadex LH-20(120 g) in methanol to give 156 mg of a mixture of thepure title compounds which are separated by silicagel chromatography(Merck 60) using 2% methanol in methylene chloride as eluant.

9'-thiaergocristine (recrystallized from benzene) m.pt. 146° (sinteringwith slow decomp); [α]_(D) ²⁰ =-176°; (c=0.5 in chloroform).

9'-thiaergocristinine (recrystallized from ethanol/ethyl acetate). m.pt.209°-210°; (decomp); [α]_(D) ²⁰ =+344° (c=0.51 in chloroform).

EXAMPLE 5

The title compounds of Example 2 may be produced in analogous manner tothat described in Example 3 or 4 using anergocryptine/eroocryptinine-producing strain.

The title compounds of Example 3 and 4 may be produced in analogousmanner to that described in Example 1 from the corresponding aminocyclolof formula II wherein either R₁ is methyl and R₂ is benzyl or R₂ isisopropyl and R₂ is benzyl.

In analogous manner to that disclosed in Example 1 the followingcompounds may be produced:

(a) 2-methyl-9,10-dihydro-9'-thia-α-ergocryptine. m.pt. 176°-180°(decomp); [α]_(D) ²⁰ =-1.25° (c=0.6 in dimethylformamide).

(b) 6-nor-6-ethyl-9,10-dihydro-9'-thia-α-ergocryptine. m.pt. 212-214°(decomp); [α]_(D) ²⁰ =+7.9° (c=0.5 in dimethylformamide).

(c) 9,10-dihydro-9'-thia-ergotamine. m.pt. 235-237°; [α]_(D) ²⁰ =-23.6°(c=0.5 in dimethylformamide).

In analogous manner to that described in Example 2 the followingcompounds are produced

(d) 2-bromo-9'-thia-α-ergocryptine.

(e) 2-bromo-9'-thia-α-ergocryptinine.

The compounds of the invention have not been described in theliterature.

The compounds of the invention are useful because they possesspharmacological activity in animals. In particular, the compounds of theinvention exhibit dopaminergic stimulant activity, as indicated bystandard animal tests.

In one test carried out according to the principles of U. Ungerstedt,Acta. physiol. Scand. Suppl. No. 367, 1971, p.69-93, 6-hydroxydopamineis injected into rats to provoke unilateral degeneration of thenigro-neo-stratal dopamine system and on injection p.o. of about 0.05 toabout 100 mg/kg (e.g. 30 to 100 mg/kg) of the compounds, the rats turncontra-laterally.

The compounds moreover induce stereotypy in the rat at a dose of about30 mg/kg i.p.

The compounds are therefore useful in the treatment of Morbus Parkinson.

For the above mentioned use the dosage will, of course, vary dependingon the compound employed, mode of administration and therapydesired.However, in general, satisfactory results are obtained whenadministered at a daily dosage of from 0.005 mg to about 100 mg per kganimal body weight, conveniently given in divided doses 2 to 4 times aday or in sustained release form. For the larger mammal, the total dailydosage is in the range from about 0.1 to about 100 mg, e.g. 0.1 to 20 mgor 1 to 100 mg, and dosage forms suitable for oral administrationcomprise from about 0.02 mg to about 50 mg of the compounds admixed witha solid or liquid pharmaceutical carrier or diluent.

Additionally, the compounds of the invention exhibit prolactin secretioninhibitory activity, as indicated by in standard tests. For example inone test effected according to the principles of Experientia 34, 1978,p. 1330 the compounds inhibit implantation in rats at a dose of fromabout 0.01 to about 1 mg/kg s.c. and inhibit lactation in rats at a doseof from about 1 to about 10 mg/kg p.o.

The compounds are therefore useful as prolactin secretion inhibitors,e.g. for the prevention or suppression of physiological lactation and totreat prolactin-induced pathological states, such as the treatment ofacromegaly.

For this use the dosage will, of course, vary depending on the compoundemployed, mode of administration and therapy desired. However, ingeneral, satisfactory results are obtained when administered at a dailydosage of from 0.01 mg to about 10 mg per kg animal bodyweight,conveniently given in divided doses 2 to 4 times a day or insustained release form. For the larger mammal, the total daily dosage isin the range from about 5 to about 100 mg, and dosage forms suitable fororal administration comprise from about 1 mg to about 50 mg of thecompounds admixed with a solid or liquid pharmaceutical carrier ordiluent.

Furthermore, the compounds exhibit anti-depressant activity, asindicated by their activity in the Ungerstedt test mentioned above andby a serotonergic effect in the sleep/wake test in the chronicallyimplanted rat at doses of from about 3 to about 10 mg/kg p.o. wherein areduction of the paradoxical sleep phase and an increase in the wakephase is observed [for method see J. M. Vigouret et al. Pharmacology,1978, 16, (1), 156-173].

The compounds are therefore useful as anti-depressant agents,particularly for old subjects.

For this use the dosage will, of course, vary depending on the compoundemployed, mode of administration and therapy desired. However, ingeneral, satisfactory results are obtained when administered at a dailydosage of from 0.1 mg to about 100 mg per kg animal body weight,conveniently given in divided doses 2 to 4 times a day or in sustainedrelease form. For the larger mammal, the total daily dosage is in therange from about 5 to about 100 mg, and dosage forms suitable for oraladministration comprise from about 1 mg to about 50 mg of the compoundsadmixed with a solid or liquid pharmaceutical carrier or diluent.

Furthermore the compounds of the invention exhibit vasoconstrictingactivity as indicated by standard tests. For example in by activity onthe Arteria carotis externa of the dog at a concentration of from about10 to about 100 nM/liter in accordance with the principles of E.Muller-Schweinitzer, Naunyn-Schmiedeberg's Arch. Pharmacol. 1976, 292,113-118.

The compounds are therefore useful as vasoconstricting agents, forexample for the treatment of migraine.

Furthermore, the compouds of the invention have venotonizing activity,as indicated by standard tests. For example in the pithed rat testeffected according to the principles of R. E. Shipley et al, Proc. Soc.exp. Bio. Med. 1947, 64, 453 or as in Brit. J. Pharmac. Chemother. 1967,30, 78-87, the compounds provoke a pressor effect and a blood pressurerise on administration of from about 5 to about 20 μg/kg i.v.Furthermore, the compounds constrict the capacitance vessels in dosesfrom about 0.5 to about 50 μg/kg in the Mellander Cat test [see formethod Angiologica, 1966, 3, 77-99].

The compounds are therefore useful as venotonizing agents e.g. for thetreatment of orthostatic hypotension, and in the prophylaxis ofthrombosis.

For the vasoconstricting and venotonizing use the dosage will, ofcourse, vary depending on the compound employed, mode of administrationand therapy desired. However, in general, satisfactory results areobtained when administered at a daily dosage of from 0.01 mg to about 10mg per kg animal body weight, conveniently given in divided doses 2 to 4times a day or in sustained release form. For the larger mammal, thetotal daily dosage is in the range from about 5 to about 100 mg, anddosage forms suitable for oral administration comprise from about 1 mgto about 50 mg of the compounds admixed with a solid or liquidpharmaceutical carrier or diluent.

Further the compounds exhibit a vigilance-increasing activity asindicated in standard tests for example in the sleep/wake test in therat as described above.

Moreover the compounds increase the local cerebral glucose utilisationin the sensomotor cartex, e.g. Hippocampus, Nucleus habenula, Nucleuscorpus gentcul . As indicated by the carbon-14-2-deoxyglucoseautoradiographic technique with the rat brain on administration i.p. offrom about 0.3 to about 30 mg/kg of the compounds [for method see e.g.L. Solokoff, Journal of Cerebral Blood Flow and Metabolism, 1981, (1),7-36, H. E. Savaki et al. Brain Research 1982, 233, 347 and J. McCullochet al. Journal of Cerebral Blood Flow and Metabolism 1981, 1, 133-136.

The compounds are therefore useful in the treatment of cerebralinsufficiency and senile dementia particularly the early stages thereof.

For this use the dosage will, of course, vary depending on the compoundemployed, mode of administration and treatment desired. However, ingeneral, satisfactory results are obtained when administered at a dailydosage of from about 0.01 mg to about 100 mg per kg animal body weight,conveniently given in divided dbses 2 to 4 times a day or in sustainedrelease form. For the larger mammal, the total daily dosage is in therange of from about 1 to about 100 e.g. 1 to 5, mg and dosage formssuitable for oral administration comprise from about 0.2 mg to about 50mg of the compounds admixed with a solid or liquid pharmaceuticalcarrier or diluent.

The present invention also provides compounds of the invention for useas dopaminergic stimulants, prolactin secretion inhibitors,anti-depressants, vasoconstricting and venotonizing agents, andvigilance increasing agents and for example for the indications definedabove.

The 9,10-dihydro-9'-thia-α-ergocryptine and2-methyl-9,10-dihydro-9'-thia-α-ergocryptine are the preferredcompounds. The vigilance increasing activity is the preferred activity.The compounds of the invention may be administered in the form of apharmaceutically acceptable acid addition salt. Such salt forms have thesame order of activity as the free base forms.

The present invention accordingly provides a compound of the inventionin free base form or in pharmaceutically acceptable acid addition saltform in association with a pharmaceutical carrier or diluent. Suchcompositions may be formulated in conventional manner so as to be, forexample, a solution or a tablet.

The compounds of the invention may be used in analogous manner tostandard compounds used for the indications mentioned above.

For example the preferred compound2-methyl-9'-thia-9,10-dihydro-α-ergocryptine exhibits in thecarbon-14-2-desoxyglucose autoradiographic test mentioned above at 3mg/kg i.p. the following increases

    ______________________________________                                                           %                                                          ______________________________________                                        Hippocampus           +2                                                      Nucleus lateralis huberula                                                                         +22                                                      Nucleus dors.corpus genicul.lat                                                                    +25                                                      ______________________________________                                    

and has a score. in the apomorphine stereotypy test of 15.7 at 30 mg/kgi.p.

What we claim is:
 1. A method of inducing a venotonizing effect in asubject which comprises administering to a subject in need of suchtreatment a venotonizing effective amount of a compound of formula I,##STR6## wherein R₁ is (C₁₋₄)alkyl,R₂ is (C₁₋₆)alkyl or benzyl, R₃ andR₄ independently are hydrogen or (C₁₋₄) alkyl, R₅ is hydrogen orbromine, R₆ and R₇ are each hydrogen, or R₆ and R₇ together form asingle bond, or R₆ is methoxy and R₇ is hydrogen, and R₈ is hydrogen,methyl, or halogen of atomic number from 9 to 35,with the proviso thatwhen R₅ is bromine, R₇ is hydrogen; in free base form or inpharmaceutically acceptable acid addition salt form.
 2. A methodaccording,to claim 1 in which the compound is9,10-dihydro-9'-thia-α-ergocryptine in free base form or inpharmaceutically acceptable acid addition salt form.
 3. A methodaccording to claim 1 in which the compound is 9'-thia-α-ergocryptinineor 9'-thia-α-ergocryptine in free base form or in pharmaceuticallyacceptable acid addition salt form.
 4. A method according to claim 1 inwhich the compound is 9'-thia-ergotamine or 9'-thia-ergotaminine in freebase form or in pharmaceutically acceptable acid addition salt form. 5.A method according to claim 1 in which the compound is9'-thiaergocristine or 9'-thiaergocristinine in free base form or inpharmaceutically acceptable acid addition salt form.
 6. A methodaccording to claim 1 in which the compound is (a)2-methyl-9,10-dihydro-9'-thia-α-ergocryptine; (b)6-nor-6-ethyl-9,10-dihydro-9'-thia-α-ergocryptine; or (c)9,10-dihydro-9'-thia-ergotamine in free base form or in pharmaceuticallyacceptable acid addition salt form.
 7. A method according to claim 1 inwhich from 0.01 mg to 10 mg per kg of animal body weight of the compoundis administered daily.
 8. A method according to claim 1 in which 5 to100 mg of the compound is administered daily.
 9. A method according toclaim 1 in which 1 to 50 mg of the compound is administered orally perunit dose.
 10. A method of treating orthostatic hypotens in a subject inneed of such treatment which comprises administering to the subject andeffective amount for the treatment of orthostatic hypotension of acompound of formula I, ##STR7## wherein R₁ is (C₁₋₄)alkyl,R₂ is(C₁₋₆)alkyl or benzyl, R₃ and R₄ independently are hydrogen or(C₁₋₄)alkyl, R₅ is hydrogen or bromine, R₆ and R₇ are each hydrogen, orR₆ and R₇ together form a single bond, or R₆ is methoxy and R₇ ishydrogen, and R₈ is hydrogen, methyl, or halogen of atomic number from 9to 35,with the proviso that when R₅ is bromine, R₇ is hydrogen; in freebase form or in pharmaceutically acceptable acid addition salt form. 11.A method according to claim 10 in which the compound is9,10-dihydro-9'-thia-α-ergocryptine in free base form or inpharmaceutically acceptable acid addition salt form.
 12. A methodaccording to claim 10 in which the compound is 9'-thia-α-ergocryptinineor 9'-thia-α-ergocryptine in free base form or in pharmaceuticallyacceptable acid addition salt form.
 13. A method according to claim 10in which the compound is 9'-thia-ergotamine or 9'-thia-ergotaminine infree base form or in pharmaceutically acceptable acid addition saltform.
 14. A method according to claim 10 in which the compound is9'-thiaergocristine or 9'-thiaergocristinine in free base form or inpharmaceutically acceptable acid addition salt form.
 15. A methodaccording to claim 10 in which the compound is (a)2-methyl-9,10-dihydro-9'thia-α-ergocryptine; (b)6-nor-6-ethyl-9,10-dihydro-9'-thia-α-ergocryptine; or (c)9,10-dihydro-9'-thia-ergotamine in free base form or in pharmaceuticallyacceptable acid addition salt form.
 16. A method according to claim 10in which from 0.01 mg to 10 mg per kg of animal body weight of thecompound is administered daily.
 17. A method according to claim 10 inwhich 5 to 100 mg of the compound is adminstered daily.
 18. A methodaccording to claim 10 in which 1 to 50 mg of the compound isadministered orally per unit dose.